Project summary Genome rearrangements are mutations that cause numerous genetic diseases including cancer, immune deficiencies and developmental disorders. Genome rearrangements are caused by defects in DNA replication and repair pathways, as well as the presence of numerous at-risk sequences in the human genome that are prone to mutations. Protein sumoylation is reversible and involves the opposing actions of two families of enzymes; the E3 ligases that catalyze the attachment of Small Ubiquitin-like MOdifier (SUMO) to substrates, and the SUMO specific proteases that remove them. Our genetic study using Saccharomyces cerevisiae has identified a major role of these enzymes in preventing diverse genome rearrangements, and has further uncovered a new paradigm in which spontaneous DNA breaks occurred during DNA replication caused these genome rearrangements. Additionally, results from our proteomic and biochemical studies have identified the Mini-Chromosome Maintenance (MCM) complex at the DNA replication fork as a major target under the regulation of SUMO. Because inherited mutations of genes in the SUMO pathways cause genome instability syndrome in mammals, understanding the molecular basis by which protein sumoylation regulates DNA replication will impact human health for two major reasons: 1) a comprehensive understanding of the genetic consequences that arise from mutations to the SUMO pathways, with regards to DNA replication, will impact the development of assays for cancer diagnosis. 2) Identifying the molecular mechanism by which SUMO regulates DNA replication will lead to the development of new therapeutic interventions of human disease. Thus, in the proposed studies we will characterize the genetics and biochemistry of the SUMO pathways in the model organism S. cerevisiae. We will pursue the following specific aims: First, we will characterize the function of protein sumoylation during chromosomal DNA replication. Second, we will investigate the role of SUMO and ubiquitin in regulating DNA replication. And finally, we will study how reversible sumoylation of the DNA replisome is regulated. The ultimate goal is to understand how SUMO regulates DNA replication and how cells utilize this pathway to prevent genome rearrangements.